尿酸是嘌呤代謝的最終產物，主要由肝臟及腸道製造，黃嘌呤氧化還原酶（XOR）以兩種形式存在，分別為黃嘌呤脫氫酶（XDH）和黃嘌呤氧化酶（XOD）。黃嘌呤氧化還原酶將次黃嘌呤代謝成黃嘌呤，再由黃嘌呤代謝為尿酸鹽/尿酸。高尿酸血症在男性中的定義為高於7 mg/dL (416 µM)，而女性為高於6 mg/dL (360 µM)。高尿酸血症被視為是一種代謝缺失異常症群，常與痛風、腎疾病、心血管疾病、糖尿病、發炎及代謝症候群有關連。因此降低血清尿酸可以預防這些慢性疾病的發生或惡化。而臨床上降尿酸藥物如: 別嘌呤醇及丙磺舒卻會有副作用如：過敏、搔癢及皮疹的產生，使這些藥物的應用受到限制，所以開發預防及治療高尿酸血症的藥物是必要的。有些微生物發酵產物因為具有抑制黃嘌呤氧化酶及尿酸酶活性進而可達到降低尿酸的效果，而紫檀芪也證實具調控尿酸運輸蛋白能力而達到降低尿酸功效。因此，在本研究中使用高尿酸血症小鼠作為動物模式及正常大鼠腎臟表皮細胞株NRK-52E探討微生物發酵產物及紫檀芪是否會降低血清中的尿酸值及抑制腎臟病變可能機轉。在動物實驗中，以管餵potassium oxonate (PO)誘發小鼠高尿酸血症的動物模式，接著管餵微生物發酵產物及紫檀芪後分析血清中生化值及肝臟黃嘌呤氧化酶 (XOD) 活性，評估是否能治療或預防高尿酸血症，接著在飼料中另外添加腺嘌呤，作為尿酸性腎病變的動物模式，探討紫檀芪是否能降低血清中的尿酸濃度、生理變化、腎功能指標、腎臟組織病理變化及纖維化相關蛋白質的表現。在細胞實驗中，以TGF-β1誘發NRK-52E細胞纖維化並預先處理紫檀芪是否能減低上皮細胞間質轉換Epithelial Mesenchymal Transition (EMT) 指標的表現，利用Trypan blue分析紫檀芪及TGF-β1對於NRK-52E細胞生長的影響，以免疫螢光及西方墨點法觀察分析細胞的型態及EMT指標的改變，最後探討可能的路徑機轉。實驗結果顯示微生物發酵產物及紫檀芪在治療模式及預防模式中能降低血清中的尿酸值，且在預防模式中能抑制肝臟XOD的活性。接著在尿酸性病變模式中，紫檀芪能降低血清中尿酸值、改善血清及尿液中CRE及BUN，在腎臟免疫組織染色中與病變組相比，能增加E-cadherin及降低Vimentin表現，並且給予紫檀芪會減少巨噬細胞的聚集以及降低TGF-β1在腎臟中表現增加。而在西方墨點法分析腎臟組織蛋白的表現，也發現給予紫檀芪能增加E-cadherin且抑制Fibronectin、Vimentin及α-SMA的表現。從NRK-52E細胞存活率結果中選出紫檀芪合適的劑量 1 µM，在可見光顯微鏡觀察下， TGF-β1誘發細胞型態變寬大，而給予紫檀芪後能減少細胞型態變寬，並從免疫螢光染色中發現給予紫檀芪處理組與TGF-β1組相比，表皮指標ZO-1增加及EMT指標Vimentin下降。在西方墨點法中，給予紫檀芪也能改善由TGF-β1誘發EMT蛋白E-cadherin、Vimentin、Fibronectin、α-SMA改變。進一步的分析自體吞噬相關蛋白的表現，發現在給予紫檀芪處理後會增加LC3-II的表現，並且在免疫螢光染色中也發現預先處理紫檀芪在加入TGF-β1比起紫檀芪和TGF-β1單獨處理處增加autophagic flux表現量，而在西方墨點法中也發現合併處比單獨處理的LC3-II的表現增加。綜合以上實驗結果，證實微生物發酵產物及紫檀芪在治療及預防模式中能透過抑制XOD表現進而降低血清中尿酸值，並在尿酸性腎病變中，紫檀芪能改善腎臟的纖維化的產生，在細胞中能改善EMT相關指標的表現， 其保護的機轉可能是透過增強自體吞噬來減低細胞纖維化。

The purpose of this study is to detect urate-lowering effects and reducing urate nephropathy mechanisms of microbial fermented extracts (MFEs) and pterostilbene (PT) by using animal and cell culture experimental models. In in vivo study, MFEs and PT hyperuricemic mice, reduced and prevented serum urate levels and liver XOD acitivity in hyperuricemic mice. In the acute toxicity study, the mice did not show any form of morbidity or mortality after gavaged MFEs. Further more, we used urate nephropathy model to confirm whether PT could prevent renal fibrosis. Our results showed that PT reduced serum urate levels and enhanced creatinine and BUN excretion in oxonate-induced hyperuricemic mice. In the kidney, HE-stained, Masson's trichrome-stained and immunohistochemistry showed PT could prevent fibrosis and EMT marker expression. Western blot analysis also demonstrated PT treatment prevent alteration in the expression of E-cadherin, Fibronectin, α-SMA and vimentin. In in vitro study, PT pretreatment could inhibite TGF-β1-induced alteration in the expression of E-cadherin, Fibronectin, α-SMA and vimentin. Moreover, PT was found to induce autophagy in NRK-52E cell, in which LC3-II expression was significantly enhanced in combined treatment with TGF-β1. Taken together, we found a new nephro-protective meachanism of PT through inhibition of TGF-β1-induced EMT by autophagy.

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